PROJECT SUMMARY Alcohol alleviates pain, but repeated exposure can produce withdrawal-induced hyperalgesia. Withdrawal- induced hyperalgesia is a pathological pain state that manifests with differing prevalence and severity in males and females, but the mechanisms that contribute to these sex differences remain unknown. Although the brain circuits that drive this pathology are not well characterized, recent evidence from the Kash Lab implicates subpopulations of midbrain and extended amygdala neurons in pain and alcohol interactions. Specifically, data from our lab has established (i) dopamine neurons in the ventrolateral periaqueductal grey (vlPAGDA+) as an alcohol-sensitive population that promotes pain relief, (ii) dorsal bed nucleus of the stria terminalis (dBNST) neurons as an alcohol-sensitive population that reduces pain, and (iii) the vlPAGDA+ projection to the dBNST as a circuit for pain reduction in males but not females. In the present proposal, we will assess sex-specific contributions of vlPAGDA+ and dBNST neurons to withdrawal-induced hyperalgesia. The first set of experiments will use slice electrophysiology and optogenetics to examine sex-specific alcohol-driven changes in vlPAGDA+ evoked transmissions to dBNST neurons. The second set of experiments will use in vivo calcium imaging to investigate whether alcohol-driven changes in pain sensitivity correlate with vlPAGDA+-driven dBNST neuronal dynamics in male and female mice. The last set of experiments will use optogenetics to selectively activate the vlPAGDA+/dBNST projection for both sexes during pain sensitivity testing, in order to assess sex differences in the pathway?s functional contributions to withdrawal-induced hyperalgesia. Taken together, the proposed experiments will strengthen our understanding of pain and alcohol interactions in the brain, providing the first sex-specific functional circuits of withdrawal-induced hyperalgesia. This knowledge will help prevent future suffering by providing novel biological targets for pain treatment in populations that excessively consume alcohol.